1 A Reproducible Method for the Quantification of Pioglitazone and Two Active Metabolites – Keto Pioglitazone and Hydroxy Pioglitazone – in Human Plasma Using Xevo TQD MS and the ACQUITY UPLC H-Class System Jennifer Simeone and Paul D. Rainville Waters Corporation, Milford, MA, USA INTRODUCTION Pioglitazone is part of the thiazolidinedione class of drugs used in the treatment of diabetes through hypoglycemic action. It selectively stimulates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ) to modulate the transcription of the insulin-sensitive genes involved in the control of glucose and lipid metabolism. 1 Following oral administration ranging from 15 to 45 mg, the dosed compound undergoes hepatic metabolism – with CYP2C8, and to a lesser degree CYP3A4, to give rise to two active metabolites; keto pioglitazone and hydroxy pioglitazone. Both metabolites are present at higher systemic concentrations than the parent compound at steady state, which is reached seven days after dosing. At steady- state, in patients with type 2 diabetes, pioglitazone comprises approximately 30% to 50% of the peak total pioglitazone serum concentrations (pioglitazone plus active metabolites) and 20% to 25% of the total AUC. In this application note, we report the development of a highly sensitive solid phase extraction, and LC/MS/MS assay using the Xevo TQD for the analysis of the pioglitazone and the two active metabolites in human plasma with an assay sensitivity of 10 pg/mL. WATERS SOLUTIONS ACQUITY UPLC H-Class System Xevo TQD MS Oasis extraction plate KEY WORDS Pioglitazone, keto pioglitazone, hydroxy pioglitazone, human plasma, hiazolidinedione, diabetes, glucose, lipid metabolism, insulin, peroxisome proliferator-activated receptor gamma, UPLC, ® Xevo TQD APPLICATION BENEFITS Waters ® Oasis ® micro-elution plates, ACQUITY UPLC ® H-Class System, and an advanced tandem quadrupole mass spectrometer (Xevo ® TQD) were used for the development of a sensitive method for quantification of pioglitazone in human plasma. This application note addresses some critical challenges in the world of bioanalysis – developing a robust and reproducible method to quantify small molecules with the desired sensitivity. Figure 1. Structure of pioglitazone (a), keto pioglitazone (b), and hydroxy pioglitazone (c).
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1
A Reproducible Method for the Quantification of Pioglitazone and Two Active Metabolites – Keto Pioglitazone and Hydroxy Pioglitazone – in Human Plasma Using Xevo TQD MS and the ACQUITY UPLC H-Class SystemJennifer Simeone and Paul D. RainvilleWaters Corporation, Milford, MA, USA
IN T RO DU C T IO N
Pioglitazone is part of the thiazolidinedione class of drugs used in the treatment
of diabetes through hypoglycemic action. It selectively stimulates the nuclear
receptor peroxisome proliferator-activated receptor gamma (PPAR-γ) to modulate
the transcription of the insulin-sensitive genes involved in the control of glucose
and lipid metabolism.1
Following oral administration ranging from 15 to 45 mg, the dosed compound
undergoes hepatic metabolism – with CYP2C8, and to a lesser degree CYP3A4,
to give rise to two active metabolites; keto pioglitazone and hydroxy pioglitazone.
Both metabolites are present at higher systemic concentrations than the parent
compound at steady state, which is reached seven days after dosing. At steady-
state, in patients with type 2 diabetes, pioglitazone comprises approximately
30% to 50% of the peak total pioglitazone serum concentrations (pioglitazone
plus active metabolites) and 20% to 25% of the total AUC.
In this application note, we report the development of a highly sensitive solid
phase extraction, and LC/MS/MS assay using the Xevo TQD for the analysis of
the pioglitazone and the two active metabolites in human plasma with an assay
sensitivity of 10 pg/mL.
WAT E R S SO LU T IO NS
ACQUITY UPLC H-Class System
Xevo TQD MS
Oasis extraction plate
K E Y W O R D S
Pioglitazone, keto pioglitazone,
hydroxy pioglitazone, human plasma,
hiazolidinedione, diabetes, glucose,
lipid metabolism, insulin, peroxisome
proliferator-activated receptor gamma,
UPLC,® Xevo TQD
A P P L I C AT IO N B E N E F I T S
Waters® Oasis® micro-elution plates,
ACQUITY UPLC® H-Class System, and an
advanced tandem quadrupole mass spectrometer
(Xevo® TQD) were used for the development
of a sensitive method for quantification of
pioglitazone in human plasma. This application
note addresses some critical challenges in the
world of bioanalysis – developing a robust and
reproducible method to quantify small molecules
with the desired sensitivity.
Figure 1. Structure of pioglitazone (a), keto pioglitazone (b), and hydroxy pioglitazone (c).
2A Reproducible Method for the Quantification of Pioglitazone and Two Active Metabolites – Keto Pioglitazone and Hydroxy Pioglitazone
SAM P L E D E S C R I P T IO N
Samples were prepared using an Oasis solid phase HLB μElution solid phase extraction plate. The plasma
samples, measuring 300 μL, were mixed with 20 μL of internal standard solution (deuturated analogues of all
three compounds) and 300 μL of 2% phosphoric acid. The samples were applied to the solid phase extraction
plate, which had previously been conditioned and equilibrated with methanol (200 μL) and water (200 μL).
The sample was washed with a 5% methanol/water solution, then eluted with a 50-μL then 25-μL aliquot of
methanol. Samples were further diluted with 75 μL of water, prior to injection.
M E T HO D CO N D I T IO NS
The analysis was performed on an ACQUITY UPLC H-Class System. A 10-μL aliquot of the sample was
injected onto an ACQUITY® BEH C18 2.1 x 50 mm, 1.7 μm column. The column was operated under gradient
conditions over 2 minutes at a flow rate of 600 μL/min. Mobile phases used were 0.1% ammonium hydroxide
and methanol. The column effluent was monitored using a Xevo TQD Mass Spectrometer operated in multiple
reaction monitoring (MRM) positive ion electrospray mode.
The transitions monitored were:
Pioglitazone: 357 > 134
Keto pioglitazone: 371 > 148
Hydroxy pioglitazone: 373 > 150
d4-pioglitazone: 361 > 138
d4-keto pioglitazone: 375 > 152
d5-hydroxy pioglitazone: 378 > 154
3A Reproducible Method for the Quantification of Pioglitazone and Two Active Metabolites – Keto Pioglitazone and Hydroxy Pioglitazone
R E SU LT S A N D D IS C U S S IO N
Pioglitazone, keto pioglitazone, and hydroxy pioglitazone eluted with retention times of 1.59, 1.35, and
1.34 minutes, respectively, as shown in Figure 2. This data shows the peaks produced by the chromatography
system are very symmetrical, and have a width at the base of approximately 3 seconds for all three compounds.
The narrow peak width, and the symmetrical nature allow for efficient processing and peak integration.
The data displayed in Figure 2 illustrates the injection of an extracted plasma blank injection, immediately
following analysis of the 1000 pg/mL standard. We can see from this data that there is no discernable
carryover in the blank chromatogram (in which the baseline has been magnified) for any of the compounds.
The extremely low carryover exhibited by the ACQUITY UPLC H-Class System allows the full sensitivity of
the Xevo TQD Mass Spectrometer to be exploited.
357 > 134 (pioglitazone)2.98e6
371 > 148 (keto pioglitazone)1.02e6
371 > 148 (keto pioglitazone)6.48e3
373 > 150 (hydroxy pioglitazone)1.11e6
373> 150 (hydroxypioglitazone)1.38e3
357 > 134 (pioglitazone)7.93e3
Figure 2. LC/MS/MS chromatogram of extracted 1000 pg/mL standard and blank for pioglitazone (a), keto pioglitazone (b), and hydroxy pioglitazone (c).
4A Reproducible Method for the Quantification of Pioglitazone and Two Active Metabolites – Keto Pioglitazone and Hydroxy Pioglitazone
The lower limit of quantification (LLOQ) for the assay was 10 pg/mL for all three analytes. Calculated signal-to-
noise values for the LLOQ were 50:1 pioglitazone/ hydroxy pioglitazone, and 22:1 keto pioglitazone, as shown in
Figure 3. A typical calibration curve obtained for the assay of pioglitazone is shown in Figure 4. The correlation
coefficient ranged between 0.997 and 0.999 for the three compounds using a 1/x weighting linear regression.
The single-day accuracy and precision data is displayed in Tables 1 through 3 for quality control (QC) samples
prepared at four levels, spanning the calibration range, and extracted in replicates of 5. The validation data shows
that the coefficient of variation for the parent and two metabolites ranged from 8.4% to 13.4% for the 10 pg/mL
LLOQ with a bias between -5.7% and 1.8%. For the high QC (800 pg/mL), the coefficient of variation ranged from
1.1% to 4.4% with a bias between -0.1% to 0.5%.
Figure 3. LLOQ (10 pg/mL) signal-to-noise values for pioglitazone (a), keto pioglitazone (b), and hydroxy pioglitazone (c).
Waters Corporation34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
CO N C LU S IO NS■■ A high sensitivity method has been developed for the analysis
of pioglitazone and its two active metabolites in human plasma
using the Xevo TQD.
■■ The assay showed excellent intra-day accuracy and precision
for QCs prepared at four concentration levels.
■■ The lower limit of quantification was determined to be
10 pg/mL with a %CV and bias, both well below the required
+/- 20% required for assay validation.
■■ The carryover was determined to be significantly less than
20% of the LLOQ in an extracted blank following the injection
of a high-concentration standard.
Waters, Xevo, Oasis, ACQUITY, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. T he Science of What’s Possible is a trademark of Waters Corporation. All other trademarks are the property of their respective owners.